a) Field of the Invention
The invention is directed to a method and an arrangement in microscopy, particularly fluorescence microscopy, for examining predominantly biological specimens, preparations and associated components. This includes methods for screening active ingredients (high throughput screening) based on fluorescence detection. Simultaneous examinations of fluorescence specimens in real time by means of simultaneous illumination of the specimen in a plurality of points on the specimen are possible.
b) Description of the Related Art
A typical area of application of light microscopy for examining biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”, Plenum Press 1995). In this case, determined dyes are used for specific labeling of cell parts.
The irradiated photons having a determined energy excite the dye molecules from the ground state to an excited state by the absorption of a photon. This excitation is usually referred to as single-photon absorption. The dye molecules excited in this way can return to the ground state in various ways. In fluorescence microscopy, the transition with emission of a fluorescence photon is most important. Because of the Stokes shift, there is generally a red shift in the wavelength of the emitted photon in comparison to the excitation radiation; that is, it has a greater wavelength. Stokes shift makes it possible to separate the fluorescence radiation from the excitation radiation.
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, several parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence).
Special dichroic beam splitters are used again to distinguish between the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of low-energy photons is also possible. The sum of energies of the single photons corresponds approximately to a multiple of the high-energy photon. This type of excitation of dyes is known as multiphoton absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996). However, the dye emission is not influenced by this type of excitation, i.e., the emission spectrum undergoes a negative Stokes shift in multiphoton absorption; that is, it has a smaller wavelength compared to the excitation radiation. The separation of the excitation radiation from the emission radiation is carried out in the same way as in single-photon excitation.
In a method according to the prior art, known as structured illumination, the modulation depth of the optical imaging of an amplitude structure (e.g., grating) is used as a criterion for the depth of field. The image of the periodic structure is distinguished by the frequency of the modulation and the phase position (image phase) of the modulation. Various projection scenarios can be obtained by means of a phase shift of the structure at right angles to the optical axis. Generally, at least three phase images PB are required at 0°, 120° and 240° to calculate depth-discriminated optical sections without stripes. These phase images (PB) are subsequently calculated to form a (confocal) optical section image in an image processor by the following formula:ISection(x)=Const•√{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}{square root over ((I(x,0°)−I(x,120°))2+(I(x,120°)−I(x,240°))2+(I(x,0°)−I(x,240°))2,)}where I(x, angle) describes the intensity at the respective pixel in the corresponding phase image.
The measuring sequence for generating an optical section image is shown schematically in FIG. 1. It is simplest to carry out the recording of three or more phase images sequentially. In this connection, it is assumed that the specimen is not moved during the measurement of the images. The section images or section stacks which are calculated in this way from the phase images can be displayed subsequently on a standard PC and monitor by means of 3-D evaluating software.
The spatial resolution along the optical axis depends on the wavelength of the light, the numerical aperture of the objective and the modulation frequency.
For a detailed description of the calculation algorithm, reference is had to T. Wilson, et al., “Method of obtaining optical sectioning by using structured light in a conventional microscope”, Optics Letters 22 (24), 1997, and WO9706509.
From a three-dimensionally illuminated image, only the plane (optical section) located in the focal plane of the objective is reproduced in connection with the corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Therefore, structured illumination is suitable for examination of thick preparations. The excitation wavelengths are determined by the utilized dye with its specific absorption characteristics. Dichroic filters adapted to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Flow cytometers are used for examining and classifying cells and other particles. For this purpose, the cells are dissolved in a liquid and are pumped through a capillary. In order to examine the cells, a laser beam is focused in the capillary from the side. The cells are dyed with different dyes or fluorescing biomolecules. The excited fluorescent light and the backscattered excitation light are measured. The separation of the fluorescence signal of the specimen from the excitation light is carried out by means of dichroic beam splitters (MDB, see FIG. 2). The art is described in “Flow Cytometry and Sorting”, 2nd edition, M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds., Wiley & Sons, Inc., New York, 1990, 81–107.
The size of the cells can be determined from the backscattered signal. Different cells can be separated and/or sorted or counted separately by means of the spectral characteristics of the fluorescence of individual cells. The sorting of the cells is carried out with an electrostatic field in different capillaries. The results, that is, for example, the quantity of cells with dye A in comparison to cells with dye B, are often displayed in histograms.
The through-flow rate is typically about 10–100 cm/s. Therefore, a highly sensitive detection is necessary. According to the prior art, a confocal detection is carried out in order to limit the detection volume.
Arrangements for screening dyes, for example, in so-called chip readers are similar in optical construction to a laser scanning microscope. However, they scan an appreciably larger image field for the examination of macroscopic specimens, for example, screening of active ingredients on a biochip. The edge length of the scan fields amounts to about 10 mm. These scan fields can be achieved, e.g., by increasing the scan angle of the galvoscanner, by arranging the specimen in an intermediate image of the microscope arrangement, for example, in FIG. 7A, or by a special objective arrangement (macroobjective) which images the intermediate image on the specimen in magnified manner.
A disadvantage in the prior art consists in that a plurality of images must be sequentially recorded, read out and calculated. In particular, this imposes increased requirements on the adjusting unit for the different projection scenarios, because otherwise residual modulations (residual structures) would remain in the image. In addition, the speed at which confocal section images can be generated is reduced by a factor of 3 when recording three phase images. Further, the usable dynamic range of the detector is limited depending on the strength of the nonconfocal background signal of the specimen (i.e., signals outside of the focal plane).